Morphine acts in vitro to directly prime nociceptors.

Hyperalgesic priming is a preclinical model of the transition from acute to chronic pain characterized by a leftward shift in the dose-response curve for and marked prolongation of prostaglandin E2 (PGE2)-induced mechanical hyperalgesia, in vivo. In vitro, priming in nociceptors is characterized by a leftward shift in the concentration dependence for PGE2-induced nociceptor sensitization. In the present in vitro study we tested the hypothesis that a mu-opioid receptor (MOR) agonist opioid analgesic, morphine, can produce priming by its direct action on nociceptors. We report that treatment of nociceptors with morphine, in vitro, produces a leftward shift in the concentration dependence for PGE2-induced nociceptor sensitization. Our findings support the suggestion that opioids act directly on nociceptors to induce priming.

The Potential Antinociceptive Effect and Mechanism of Cannabis sativa L. Extract on Paclitaxel-Induced Neuropathic Pain in Rats Uncovered by Multi-Omics Analysis.

Cannabis sativa L. (hemp) is a herbaceous plant rich in cannabinoids with a long history of use in pain treatment. The most well-characterized cannabinoids, cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC), garnered much attention in chemotherapy-induced peripheral neuropathy (CIPN) treatment. However, few studies have investigated the biological benefits and mechanism of hemp extract on CIPN. In the present study, hemp extract (JG) rich in cannabinoids was extracted by supercritical fluid carbon dioxide extraction (SFCE). The antinociceptive efficacy was evaluated using a paclitaxel-induced peripheral neuropathy (PIPN) rat model based on behavioral tests. Further omics-based approaches were applied to explore the potential mechanisms. The results showed that JG decreased mechanical allodynia, thermal hyperalgesia, and inflammatory cytokines in PIPN rats significantly. Transcriptome analysis identified seven key genes significantly regulated by JG in PIPN model rats, mainly related to the neuroactive ligand-receptor interaction pathway, PPAR signaling pathway, and cAMP signaling pathway. In metabolomic analysis, a total of 39 significantly altered metabolites were identified, mainly correlated with pentose and glucuronate interconversions and the glycerophospholipid metabolism pathway. Gut microbiota analysis suggested that increased community Lachnoclostridium and Lachnospiraceae_UCG-006 in PIPN rats can be reversed significantly by JG. In conclusion, hemp extract exhibited antinociceptive effects on PIPN. The analgesic mechanism was probably related to the regulation of inflammation, neuroactive ligand-receptor interaction pathway, sphingolipid metabolism, etc. This study provides novel insights into the functional interactions of Cannabis sativa L. extract on PIPN.

Contribution of mechanoreceptors to spinal cord injury-induced mechanical allodynia.

ABSTRACT: Evidence from previous studies supports the concept that spinal cord injury (SCI)-induced neuropathic pain (NP) has its neural roots in the peripheral nervous system. There is uncertainty about how and to which degree mechanoreceptors contribute. Sensorimotor activation-based interventions (eg, treadmill training) have been shown to reduce NP after experimental SCI, suggesting transmission of pain-alleviating signals through mechanoreceptors. The aim of the present study was to understand the contribution of mechanoreceptors with respect to mechanical allodynia in a moderate mouse contusion SCI model. After genetic ablation of tropomyosin receptor kinase B expressing mechanoreceptors before SCI, mechanical allodynia was reduced. The identical genetic ablation after SCI did not yield any change in pain behavior. Peptidergic nociceptor sprouting into lamina III/IV below injury level as a consequence of SCI was not altered by either mechanoreceptor ablation. However, skin-nerve preparations of contusion SCI mice 7 days after injury yielded hyperexcitability in nociceptors, not in mechanoreceptors, which makes a substantial direct contribution of mechanoreceptors to NP maintenance unlikely. Complementing animal data, quantitative sensory testing in human SCI subjects indicated reduced mechanical pain thresholds, whereas the mechanical detection threshold was not altered. Taken together, early mechanoreceptor ablation modulates pain behavior, most likely through indirect mechanisms. Hyperexcitable nociceptors seem to be the main drivers of SCI-induced NP. Future studies need to focus on injury-derived factors triggering early-onset nociceptor hyperexcitability, which could serve as targets for more effective therapeutic interventions.

Environmental enrichment alleviates hyperalgesia by modulating central sensitization in a nitroglycerin-induced chronic migraine model of mice.

BACKGROUND: Chronic migraine (CM) is a debilitating neurofunctional disorder primarily affecting females, characterized by central sensitization. Central sensitization refers to the enhanced response to sensory stimulation, which involves changes in neuronal excitability, synaptic plasticity, and neurotransmitter release. Environmental enrichment (EE) can increase the movement, exploration, socialization and other behaviors of mice. EE has shown promising effects in various neurological disorders, but its impact on CM and the underlying mechanism remains poorly understood. Therefore, the purpose of this study was to determine whether EE has the potential to serve as a cost-effective intervention strategy for CM. METHODS: A mouse CM model was successfully established by repeated administration of nitroglycerin (NTG). We selected adult female mice around 8 weeks old, exposed them to EE for 2 months, and then induced the CM model. Nociceptive threshold tests were measured using Von Frey filaments and a hot plate. The expression of c-Fos, calcitonin gene-related peptide (CGRP) and inflammatory response were measured using WB and immunofluorescence to evaluate central sensitization. RNA sequencing was used to find differentially expressed genes and signaling pathways. Finally, the expression of the target differential gene was investigated. RESULTS: Repeated administration of NTG can induce hyperalgesia in female mice and increase the expression of c-Fos and CGRP in the trigeminal nucleus caudalis (TNC). Early exposure of mice to EE reduced NTG-induced hyperalgesia in CM mice. WB and immunofluorescence revealed that EE inhibited the overexpression of c-Fos and CGRP in the TNC of CM mice and alleviated the inflammatory response of microglia activation. RNA sequencing analysis identified that several central sensitization-related signaling pathways were altered by EE. VGluT1, a key gene involved in behavior, internal stimulus response, and ion channel activity, was found to be downregulated in mice exposed to EE. CONCLUSION: EE can significantly ameliorate hyperalgesia in the NTG-induced CM model. The mechanisms may be to modulate central sensitization by reducing the expression of CGRP, attenuating the inflammatory response, and downregulating the expression of VGluT1, etc., suggesting that EE can serve as an effective preventive strategy for CM.

Extrasynaptic δGABAA receptors mediate resistance to migraine-like phenotype in rats.

BACKGROUND: GABA, a key inhibitory neurotransmitter, has synaptic and extrasynaptic receptors on the postsynaptic neuron. Background GABA, which spills over from the synaptic cleft, acts on extrasynaptic delta subunit containing GABAA receptors. The role of extrasynaptic GABAergic input in migraine is unknown. We investigated the susceptibility to valid migraine-provoking substances with clinically relevant behavioral readouts in Genetic Absence Epilepsy of Rats Strasbourg (GAERS), in which the GABAergic tonus was altered. Subsequently, we screened relevant GABAergic mechanisms in Wistar rats by pharmacological means to identify the mechanisms. METHODS: Wistar and GAERS rats were administered nitroglycerin (10 mg/kg) or levcromakalim (1 mg/kg). Mechanical allodynia and photophobia were assessed using von Frey monofilaments and a dark-light box. Effects of GAT-1 blocker tiagabine (5 mg/kg), GABAB receptor agonist baclofen (2 mg/kg), synaptic GABAA receptor agonist diazepam (1 mg/kg), extrasynaptic GABAA receptor agonists gaboxadol (4 mg/kg), and muscimol (0.75 mg/kg), T-type calcium channel blocker ethosuximide (100 mg/kg) or synaptic GABAA receptor antagonist flumazenil (15 mg/kg) on levcromakalim-induced migraine phenotype were screened. RESULTS: Unlike Wistar rats, GAERS exhibited no reduction in mechanical pain thresholds or light aversion following nitroglycerin or levcromakalim injection. Ethosuximide did not reverse the resistant phenotype in GAERS, excluding the role of T-type calcium channel dysfunction in this phenomenon. Tiagabine prevented levcromakalim-induced mechanical allodynia in Wistar rats, suggesting a key role in enhanced GABA spillover. Baclofen did not alleviate mechanical allodynia. Diazepam failed to mitigate levcromakalim-induced migraine phenotype. Additionally, the resistant phenotype in GAERS was not affected by flumazenil. Extrasynaptic GABAA receptor agonists gaboxadol and muscimol inhibited periorbital allodynia in Wistar rats. CONCLUSION: Our study introduced a rat strain resistant to migraine-provoking agents and signified a critical involvement of extrasynaptic δGABAergic receptors. Extrasynaptic δ GABAA receptors, by mediating constant background inhibition on the excitability of neurons, stand as a novel drug target with a therapeutic potential in migraine.

Effects of the Dual FAAH/MAGL Inhibitor AKU-005 on Trigeminal Hyperalgesia in Male Rats.

The inhibition of endocannabinoid hydrolysis by enzymatic inhibitors may interfere with mechanisms underlying migraine-related pain. The dual FAAH/MAGL inhibitor AKU-005 shows potent inhibitory activity in vitro. Here, we assessed the effect of AKU-005 in a migraine animal model based on nitroglycerin (NTG) administration. Male rats were treated with AKU-005 (0.5 mg/kg, i.p.) or vehicle 3 h after receiving NTG (10 mg/kg, i.p.) or NTG vehicle. One hour later, rats were subjected to the open field test followed by the orofacial formalin test. At the end of the test, we collected serum samples for assessing calcitonin gene-related peptide (CGRP) levels as well as meninges, trigeminal ganglia, and brain areas to assess mRNA levels of CGRP and pro-inflammatory cytokines, and endocannabinoid and related lipid levels. AKU-005 reduced NTG-induced hyperalgesia during the orofacial formalin test but did not influence NTG-induced changes in the open field test. It significantly reduced serum levels of CGRP, CGRP, and pro-inflammatory cytokine mRNA levels in the meninges, trigeminal ganglia, and central areas. Surprisingly, AKU-005 caused no change in endocannabinoids and related lipids in the regions evaluated. The present findings suggest that AKU-005 may have anti-migraine effects by reducing CGRP synthesis and release and the associated inflammatory events. This effect, however, does not seem mediated via an interference with the endocannabinoid pathway.

Reduced sympathetic activity is associated with the development of pain and muscle atrophy in a female rat model of fibromyalgia.

Fibromyalgia (FM) is characterized by chronic widespread musculoskeletal pain accompanied by fatigue and muscle atrophy. Although its etiology is not known, studies have shown that FM patients exhibit altered function of the sympathetic nervous system (SNS), which regulates nociception and muscle plasticity. Nevertheless, the precise SNS-mediated mechanisms governing hyperalgesia and skeletal muscle atrophy in FM remain unclear. Thus, we employed two distinct FM-like pain models, involving intramuscular injections of acidic saline (pH 4.0) or carrageenan in prepubertal female rats, and evaluated the catecholamine content, adrenergic signaling and overall muscle proteolysis. Subsequently, we assessed the contribution of the SNS to the development of hyperalgesia and muscle atrophy in acidic saline-injected rats treated with clenbuterol (a selective ß2-adrenergic receptor agonist) and in animals maintained under baseline conditions and subjected to epinephrine depletion through adrenodemedullation (ADM). Seven days after inducing an FM-like model with acidic saline or carrageenan, we observed widespread mechanical hyperalgesia along with loss of strength and/or muscle mass. These changes were associated with reduced catecholamine content, suggesting a common underlying mechanism. Notably, treatment with a ß2-agonist alleviated hyperalgesia and prevented muscle atrophy in acidic saline-induced FM-like pain, while epinephrine depletion induced mechanical hyperalgesia and increased muscle proteolysis in animals under baseline conditions. Together, the results suggest that reduced sympathetic activity is involved in the development of pain and muscle atrophy in the murine model of FM analyzed.

Dissecting Genetic Mechanisms of Differential Locomotion, Depression, and Allodynia after Spinal Cord Injury in Three Mouse Strains.

Strain differences have been reported for motor behaviors, and only a subset of spinal cord injury (SCI) patients develop neuropathic pain, implicating genetic or genomic contribution to this condition. Here, we evaluated neuropsychiatric behaviors in A/J, BALB/c, and C57BL/6 male mice and tested genetic or genomic alterations following SCI. A/J and BALB/c naive mice showed significantly less locomotor activity and greater anxiety-like behavior than C57BL/6 mice. Although SCI elicited locomotor dysfunction, C57BL/6 and A/J mice showed the best and the worst post-traumatic recovery, respectively. Mild (m)-SCI mice showed deficits in gait dynamics. All moderate/severe SCI mice exhibited similar degrees of anxiety/depression. mSCI in BALB/c and A/J mice resulted in depression, whereas C57BL/6 mice did not exhibit depression. mSCI mice had significantly lower mechanical thresholds than their controls, indicating high cutaneous hypersensitivity. C57BL/6, but not A/J and BLAB/c mice, showed significantly lower heat thresholds than their controls. C57BL/6 mice exhibited spontaneous pain. RNAseq showed that genes in immune responses and wound healing were upregulated, although A/J mice showed the largest increase. The cell cycle and the truncated isoform of trkB genes were robustly elevated in SCI mice. Thus, different genomics are associated with post-traumatic recovery, underscoring the likely importance of genetic factors in SCI.

Electroacupuncture attenuates neuropathic pain via suppressing BIP-IRE-1α-mediated endoplasmic reticulum stress in the anterior cingulate cortex.

Studies have suggested that endoplasmic reticulum stress (ERS) is involved in neurological dysfunction and that electroacupuncture (EA) attenuates neuropathic pain (NP) via undefined pathways. However, the role of ERS in the anterior cingulate cortex (ACC) in NP and the effect of EA on ERS in the ACC have not yet been investigated. In this study, an NP model was established by chronic constriction injury (CCI) of the left sciatic nerve in rats, and mechanical and cold tests were used to evaluate behavioral hyperalgesia. The protein expression and distribution were evaluated using western blotting and immunofluorescence. The results showed that glucose-regulated protein 78 (BIP) and inositol-requiring enzyme 1α (IRE-1α) were co-localized in neurons in the ACC. After CCI, BIP, IRE-1α, and phosphorylation of IRE-1α were upregulated in the ACC. Intra-ACC administration of 4-PBA and Kira-6 attenuated pain hypersensitivity and downregulated phosphorylation of IRE-1α, while intraperitoneal injection of 4-PBA attenuated hyperalgesia and inhibited the activation of P38 and JNK in ACC. In contrast, ERS activation by intraperitoneal injection of tunicamycin induced behavioral hyperalgesia in naive rats. Furthermore, EA attenuated pain hypersensitivity and inhibited the CCI-induced overexpression of BIP and pIRE-1α. Taken together, these results demonstrate that EA attenuates NP by suppressing BIP- and IRE-1α-mediated ERS in the ACC. Our study presents novel evidence that ERS in the ACC is implicated in the development of NP and provides insights into the molecular mechanisms involved in the analgesic effect of EA.

Electroacupuncture improves allodynia and central sensitization via modulation of microglial activation associated P2X4R and inflammation in a rat model of migraine.

Background: Recent studies have demonstrated that activated microglia were involved in the pathogenesis of central sensitization characterized by cutaneous allodynia in migraine. Activation of microglia is accompanied by increased expression of its receptors and release of inflammatory mediators. Acupuncture and its developed electroacupuncture (EA) have been recommended as an alternative therapy for migraine and are widely used for relieving migraine-associated pain. However, it remains rare studies that show whether EA exerts anti-migraine effects via inhibiting microglial activation related to a release of microglial receptors and the inflammatory pathway. Therefore, this study aimed to investigate EA' ability to ameliorate central sensitization via modulation of microglial activation, microglial receptor, and inflammatory response using a rat model of migraine induced by repeated epidural chemical stimulation. Methods: In the present study, a rat model of migraine was established by epidural repeated inflammatory soup (IS) stimulation and treated with EA at Fengchi (GB20) and Yanglingquan (GB34) and acupuncture at sham-acupoints. Pain hypersensitivity was further determined by measuring the mechanical withdrawal threshold using the von-Frey filament. The changes in c-Fos and ionized calcium binding adaptor molecule 1 (Ibal-1) labeled microglia in the trigeminal nucleus caudalis (TNC) were examined by immunflurescence to assess the central sensitization and whether accompanied with microglia activation. In addition, the expression of Ibal-1, microglial purinoceptor P2X4, and its associated inflammatory signaling pathway mediators, including interleukin (IL)-1ß, NOD-like receptor protein 3 (NLRP3), and Caspase-1 in the TNC were investigated by western blot and real-time polymerase chain reaction analysis. Results: Allodynia increased of c-Fos, and activated microglia were observed after repeated IS stimulation. EA alleviated the decrease in mechanical withdrawal thresholds, reduced the activation of c-Fos and microglia labeled with Ibal-1, downregulated the level of microglial purinoceptor P2X4, and limited the inflammatory response (NLRP3/Caspase-1/IL-1ß signaling pathway) in the TNC of migraine rat model. Conclusions: Our results indicate that the anti-hyperalgesia effects of EA ameliorate central sensitization in IS-induced migraine by regulating microglial activation related to P2X4R and NLRP3/IL-1ß inflammatory pathway.

Early evidence of beneficial and protective effects of Protectin DX treatment on behavior responses and type-1 diabetes mellitus related-parameters: A non-clinical approach.

Protectin DX (PDX), a specialized pro-resolving lipid mediator, presents potential therapeutic applications across various medical conditions due to its anti-inflammatory and antioxidant properties. Since type-1 diabetes mellitus (T1DM) is a disease with an inflammatory and oxidative profile, exploring the use of PDX in addressing T1DM and its associated comorbidities, including diabetic neuropathic pain, depression, and anxiety becomes urgent. Thus, in the current study, after 2 weeks of T1DM induction with streptozotocin (60 mg/kg) in Wistar rats, PDX (1, 3, and 10 ng/animal; i.p. injection of 200 µl/animal) was administered specifically on days 14, 15, 18, 21, 24, and 27 after T1DM induction. We investigated the PDX's effectiveness in alleviating neuropathic pain (mechanical allodynia; experiment 1), anxiety-like and depressive-like behaviors (experiment 2). Also, we studied whether the PDX treatment would induce antioxidant effects in the blood plasma, hippocampus, and prefrontal cortex (experiment 3), brain areas involved in the modulation of emotions. For evaluating mechanical allodynia, animals were repeatedly submitted to the Von Frey test; while for studying anxiety-like responses, animals were submitted to the elevated plus maze (day 26) and open field (day 28) tests. To analyze depressive-like behaviors, the animals were tested in the modified forced swimming test (day 28) immediately after the open field test. Our data demonstrated that PDX consistently increased the mechanical threshold throughout the study at the two highest doses, indicative of antinociceptive effect. Concerning depressive-like and anxiety-like behavior, all PDX doses effectively prevented these behaviors when compared to vehicle-treated T1DM rats. The PDX treatment significantly protected against the increased oxidative stress parameters in blood plasma and in hippocampus and prefrontal cortex. Interestingly, treated animals presented improvement on diabetes-related parameters by promoting weight gain and reducing hyperglycemia in T1DM rats. These findings suggest that PDX improved diabetic neuropathic pain, and induced antidepressant-like and anxiolytic-like effects, in addition to improving parameters related to the diabetic condition. It is worth noting that PDX also presented a protective action demonstrated by its antioxidant effects. To conclude, our findings suggest PDX treatment may be a promising candidate for improving the diabetic condition per se along with highly disabling comorbidities such as diabetic neuropathic pain and emotional disturbances associated with T1DM.

Tonic excitation by astrocytic GABA causes neuropathic pain by augmenting neuronal activity and glucose metabolism.

Neuropathic pain is a debilitating condition caused by the hyperexcitability of spinal dorsal horn neurons and is often characterized by allodynia. Although neuron-independent mechanisms of hyperexcitability have been investigated, the contribution of astrocyte-neuron interactions remains unclear. Here, we show evidence of reactive astrocytes and their excessive GABA release in the spinal dorsal horn, which paradoxically leads to the tonic excitation of neighboring neurons in a neuropathic pain model. Using multiple electrophysiological methods, we demonstrated that neuronal hyperexcitability is attributed to both increased astrocytic GABA synthesis via monoamine oxidase B (MAOB) and the depolarized reversal potential of GABA-mediated currents (EGABA) via the downregulation of the neuronal K+/Cl- cotransporter KCC2. Furthermore, longitudinal 2-deoxy-2-[18F]-fluoro-D-glucose microPET imaging demonstrated increased regional glucose metabolism in the ipsilateral dorsal horn, reflecting neuronal hyperexcitability. Importantly, inhibiting MAOB restored the entire astrocytic GABA-mediated cascade and abrogated the increased glucose metabolism and mechanical allodynia. Overall, astrocytic GABA-mediated tonic excitation is critical for neuronal hyperexcitability, leading to mechanical allodynia and neuropathic pain.

Common alterations in parallel metabolomic profiling of serum and spinal cord and mechanistic studies on neuropathic pain following PPARα administration.

Neuropathic pain (NP) is usually treated with analgesics and symptomatic therapy with poor efficacy and numerous side effects, highlighting the urgent need for effective treatment strategies. Recent studies have reported an important role for peroxisome proliferator-activated receptor alpha (PPARα) in regulating metabolism as well as inflammatory responses. Through pain behavioral assessment, we found that activation of PPARα prevented chronic constriction injury (CCI)-induced mechanical allodynia and thermal hyperalgesia. In addition, PPARα ameliorated inflammatory cell infiltration at the injury site and decreased microglial activation, NOD-like receptor protein 3 (NLRP3) inflammasome production, and spinal dendritic spine density, as well as improved serum and spinal cord metabolic levels in mice. Administration of PPARα antagonists eliminates the analgesic effect of PPARα agonists. PPARα relieves NP by inhibiting neuroinflammation and functional synaptic plasticity as well as modulating metabolic mechanisms, suggesting that PPARα may be a potential molecular target for NP alleviation. However, the effects of PPARα on neuroinflammation and synaptic plasticity should be further explored.

Photobiomodulation improves acute restraint stress-induced visceral hyperalgesia in rats.

The purpose of this study is to explore the potential application of photobiomodulation to irritable bowel syndrome. We established the following experimental groups: the Non-Stress + Sham group, which consisted of rats that were not restrained and were only subjected to sham irradiation; the Stress + Sham group, which underwent 1 hour of restraint stress followed by sham irradiation; and the Stress + Laser group, which was subjected to restraint stress and percutaneous laser irradiation bilaterally on the L6 dorsal root ganglia for 5 minutes each. The experiment was conducted twice, with three and two laser conditions examined. Following laser irradiation, a barostat catheter was inserted into the rat's colon. After a 30-minute acclimatization period, the catheter was inflated to a pressure of 60 mmHg, and the number of abdominal muscle contractions was measured over a 5-minute period. The results showed that photobiomodulation significantly suppressed the number of abdominal muscle contractions at average powers of 460, 70, and 18 mW. However, no significant suppression was observed at average powers of 1 W and 3.5 mW. This study suggests that photobiomodulation can alleviate visceral hyperalgesia induced by restraint stress, indicating its potential applicability to irritable bowel syndrome.

Novel Scorpion Toxin ω-Buthitoxin-Hf1a Selectively Inhibits Calcium Influx via CaV3.3 and CaV3.2 and Alleviates Allodynia in a Mouse Model of Acute Postsurgical Pain.

Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 µM) and CaV3.3 (IC50 = 0.49 µM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.

Efficacy assessment of novel methanimine derivatives in chronic constriction injury-induced neuropathic model: An in-vivo, ex-vivo and In-Silico approach.

The multicomponent etiology, complex clinical implications, dose-based side effect and degree of pain mitigation associated with the current pharmacological therapy is incapable in complete resolution of chronic neuropathic pain patients which necessitates the perpetual requirement of novel medication therapy. Therefore, this study explored the ameliorative aptitude of two novel methanimine imitative like (E)-N-(4-nitrobenzylidene)-4­chloro-2-iodobenzamine (KB 09) and (E)-N-(4-methylbenzylidene)-4­chloro-2-iodobenzamine (KB 10) in chronic constriction injury (CCI) of sciatic nerve induced neuropathic pain in rat model. Standard behavioral tests like dynamic and static allodynia, cold, thermal and mechanical hyperalgesia along with rotarod activity were performed at various experimental days like 0, 3, 7, 14 and 21. Enzyme linked immunosorbent assay (ELISA) on spinal tissue and antioxidant assays on sciatic nerve were executed accompanied by molecular docking and simulation studies. Prolonged ligation of sciatic nerve expressively induced hyperalgesia as well as allodynia in rats. KB 09 and KB 10 substantially attenuated the CCI elicited hyperalgesia and allodynia. They significantly reduced the biomarkers of pain and inflammation like Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in ELISA and while enhanced the GSH, SOD and CAT and diminished the MDA levels during antioxidant assays. KB 09 displayed -9.62 kcal/mol with TNF-α and -7.68 kcal/mol binding energy with IL-6 whereas KB 10 exhibited binding energy of -8.20 kcal/mol with IL-6 while -11.68 kcal/mol with TNF-α and hence both trial compounds ensured stable interaction with IL-6 and TNF-α during computational analysis. The results advocated that both methanimine derivatives might be novel candidates for attenuation of CCI-induced neuropathic pain prospects via anti-nociceptive, anti-inflammatory and antioxidant mechanisms.

Plp1-expresssing perineuronal DRG cells facilitate colonic and somatic chronic mechanical pain involving Piezo2 upregulation in DRG neurons.

Satellite glial cells (SGCs) of dorsal root ganglia (DRGs) are activated in a variety of chronic pain conditions; however, their mediation roles in pain remain elusive. Here, we take advantage of proteolipid protein (PLP)/creERT-driven recombination in the periphery mainly occurring in SGCs of DRGs to assess the role of SGCs in the regulation of chronic mechanical hypersensitivity and pain-like responses in two organs, the distal colon and hindpaw, to test generality. We show that PLP/creERT-driven hM3Dq activation increases, and PLP/creERT-driven TrkB.T1 deletion attenuates, colon and hindpaw chronic mechanical hypersensitivity, positively associating with calcitonin gene-related peptide (CGRP) expression in DRGs and phospho-cAMP response element-binding protein (CREB) expression in the dorsal horn of the spinal cord. Activation of Plp1+ DRG cells also increases the number of small DRG neurons expressing Piezo2 and acquiring mechanosensitivity and leads to peripheral organ neurogenic inflammation. These findings unravel a role and mechanism of Plp1+ cells, mainly SGCs, in the facilitation of chronic mechanical pain and suggest therapeutic targets for pain mitigation.

Exploration on pharmacological mechanisms of YZP against neuropathic pain via inhibiting spinal inflammation and the rationality of its compatibility.

ETHNOPHARMACOLOGICAL RELEVANCE: Yuanhu Zhitong Prescription (YZP) is a well-known traditional Chinese medicine (TCM) formula for neuropathic pain (NP) therapy with a satisfying clinical efficacy. However, the underlying pharmacological mechanism and its compatibility principle remain unclear. AIM OF THE STUDY: This study aims to investigate the analgesic and compatibility mechanisms of YZP on neuropathic pain (NP) at the gene and biological process levels. MATERIALS AND METHODS: The chronic constriction injury (CCI) rats were intragastrically administrated with extracts of YZP, YH and BZ separately, and then mechanical hypersensitivity were measured to evaluate the analgesic effects between YH and BZ before and after compatibility. Then, RNA-seq and bioinformatics analyses were performed to elucidate the potential mechanisms underlying YZP's analgesia and compatibility. Finally, the expression levels and significant differences of key genes were analyzed. RESULTS: Behaviorally, both YZP and YH effectively alleviated mechanical allodynia in CCI rats, with YZP being superior to YH. In contrast, we did not observe an analgesic effect of BZ. Genetically, YZP, YH, and BZ reversed the expression levels of 52, 34, and 42 aberrant genes in the spinal cord of CCI rats, respectively. Mechanically, YZP was revealed to alleviate NP mainly by modulating the inflammatory response and neuropeptide signaling pathway, which are the dominant effective processes of YH. Interestingly, the effective targets of YZP were especially enriched in leukocyte activation and cytokine-mediated signaling pathways. Moreover, BZ was found to exert an adjunctive effect in enhancing the analgesic effect of YH by promoting skeletal muscle tissue regeneration and modulating calcium ion transport. CONCLUSIONS: YH, as the monarch drug, plays a dominant role in the analgesic effect of YZP that effectively relieves NP by inhibiting the spinal inflammation and neuropeptide signaling pathway. BZ, as the minister drug, not only synergistically enhances analgesic processes of YH but also helps to alleviate the accompanying symptoms of NP. Consequently, YZP exerted a more potent analgesic effect than YH and BZ alone. In conclusion, our findings offer new insights into understanding the pharmacological mechanism and compatibility principle of YZP, which may support its clinical application in NP therapy.

Glucose competition between endothelial cells in the blood-spinal cord barrier and infiltrating regulatory T cells is linked to sleep restriction-induced hyperalgesia.

BACKGROUND: Sleep loss is a common public health problem that causes hyperalgesia, especially that after surgery, which reduces the quality of life seriously. METHODS: The 48-h sleep restriction (SR) mouse model was created using restriction chambers. In vivo imaging, transmission electron microscopy (TEM), immunofluorescence staining and Western blot were performed to detect the status of the blood-spinal cord barrier (BSCB). Paw withdrawal mechanical threshold (PWMT) was measured to track mouse pain behavior. The role of infiltrating regulatory T cells (Tregs) and endothelial cells (ECs) in mouse glycolysis and BSCB damage were analyzed using flow cytometry, Western blot, CCK-8 assay, colorimetric method and lactate administration. RESULTS: The 48-h SR made mice in sleep disruption status and caused an acute damage to the BSCB, resulting in hyperalgesia and neuroinflammation in the spinal cord. In SR mice, the levels of glycolysis and glycolysis enzymes of ECs in the BSCB were found significantly decreased [CON group vs. SR group: CD31+Glut1+ cells: p < 0.001], which could cause dysfunction of ECs and this was confirmed in vitro. Increased numbers of infiltrating T cells [p < 0.0001] and Treg population [p < 0.05] were detected in the mouse spinal cord after 48-h SR. In the co-cultured system of ECs and Tregs in vitro, the competition of Tregs for glucose resulted in the glycolysis disorder of ECs [Glut1: p < 0.01, ENO1: p < 0.05, LDHα: p < 0.05; complete tubular structures formed: p < 0.0001; CCK8 assay: p < 0.001 on 24h, p < 0.0001 on 48h; glycolysis level: p < 0.0001]. An administration of sodium lactate partially rescued the function of ECs and relieved SR-induced hyperalgesia. Furthermore, the mTOR signaling pathway was excessively activated in ECs after SR in vivo and those under the inhibition of glycolysis or co-cultured with Tregs in vitro. CONCLUSIONS: Affected by glycolysis disorders of ECs due to glucose competition with infiltrating Tregs through regulating the mTOR signaling pathway, hyperalgesia induced by 48-h SR is attributed to neuroinflammation and damages to the barriers, which can be relieved by lactate supplementation.

Implication of system xc- in neuroinflammation during the onset and maintenance of neuropathic pain.

BACKGROUND: Despite the high prevalence of neuropathic pain, treating this neurological disease remains challenging, given the limited efficacy and numerous side effects associated with current therapies. The complexity in patient management is largely attributed to an incomplete understanding of the underlying pathological mechanisms. Central sensitization, that refers to the adaptation of the central nervous system to persistent inflammation and heightened excitatory transmission within pain pathways, stands as a significant contributor to persistent pain. Considering the role of the cystine/glutamate exchanger (also designated as system xc-) in modulating glutamate transmission and in supporting neuroinflammatory responses, we investigated the contribution of this exchanger in the development of neuropathic pain. METHODS: We examined the implication of system xc- by evaluating changes in the expression/activity of this exchanger in the dorsal spinal cord of mice after unilateral partial sciatic nerve ligation. In this surgical model of neuropathic pain, we also examined the consequence of the genetic suppression of system xc- (using mice lacking the system xc- specific subunit xCT) or its pharmacological manipulation (using the pharmacological inhibitor sulfasalazine) on the pain-associated behavioral responses. Finally, we assessed the glial activation and the inflammatory response in the spinal cord by measuring mRNA and protein levels of GFAP and selected M1 and M2 microglial markers. RESULTS: The sciatic nerve lesion was found to upregulate system xc- at the spinal level. The genetic deletion of xCT attenuated both the amplitude and the duration of the pain sensitization after nerve surgery, as evidenced by reduced responses to mechanical and thermal stimuli, and this was accompanied by reduced glial activation. Consistently, pharmacological inhibition of system xc- had an analgesic effect in lesioned mice. CONCLUSION: Together, these observations provide evidence for a role of system xc- in the biochemical processes underlying central sensitization. We propose that the reduced hypersensitivity observed in the transgenic mice lacking xCT or in sulfasalazine-treated mice is mediated by a reduced gliosis in the lumbar spinal cord and/or a shift in microglial M1/M2 polarization towards an anti-inflammatory phenotype in the absence of system xc-. These findings suggest that drugs targeting system xc- could contribute to prevent or reduce neuropathic pain.